Polypeptides selectively reactive with antibodies against human immunodeficiency virus and vaccines comprising the polypeptides

ABSTRACT

Highly conserved polypeptide sequences derived from gp41 and gp120, preferably from eleven to twenty-one amino acids in length, are joined (for example, via DNA recombinant techniques) to a non-HIV protein or polypeptide sequence comprising an amino-acid sequence not naturally encoded by the HIV genome, thereby forming a fusion protein. Such fusion proteins possess attributes that make them suitable for use in the diagnosis, treatment and prevention of HIV infection.

BACKGROUND OF THE INVENTION

This invention was made in part with U.S. Government Support underContract No. DAMD17-87-C-7156 awarded by the United States Army MedicalResearch and Development Command to the National Research Council. TheU.S. Government has certain rights in this invention.

The present invention relates to the production and use, in diagnosis,treatment and prevention of HIV infection, of an antigenic protein thatreacts specifically with antibodies against the Human ImmunodeficiencyVirus type 1 (HIV-1). The present invention also relates to aself-replicating cell that produces such a recombinant protein; to avaccine comprising the recombinant protein; to an antigen-basedscreening test, based on the protein, for detecting antibodies to HIV;to monoclonal and polyclonal antibodies, produced using the protein,that protect against HIV-1 infection or disease; and to anantibody-based screening test.

HIV has been established as the primary etiologic agent in pathogenesisof acquired immunodeficiency syndrome (AIDS) and related disorders. See,e.g., Gruest, J., et al., Science 220: 863-71 (1983); Gallo, R. C., etal., Science 224: 500-03 (1984). Because AIDS can be transmitted byblood products, a highly accurate method of screening blood samples forpresence of the virus is desirable. Infection of humans with HIV leadsto production of antibodies directed against most of the viralstructural antigens, forming the basis for screening via viral lysatebased tests. However, these tests have several disadvantages, includingfalse positives thought to arise from the presence of non-viral proteinsin the viral lysate preparations used in the solid phase component ofthe current assays.

The antibodies produced upon infection include antibodies against bothcore and envelope proteins. The emergence of antibodies to envelopeglycoproteins, e.g., gp160, and its subunits gp120 (the extracellularglycoprotein or EGP) and gp41 (the transmembrane protein or TMP),appears to precede the emergence of antibodies to core proteins, leadingresearchers to study these proteins as a possible basis for improveddiagnostic assays. In addition, these antibodies to env proteins appearto be involved in induction of active immunity, suggesting their use invaccine preparation.

Based on study of the envelope amino acid sequences, and in an attemptto reduce the rate of false positives, the art has proposed serologicalassays employing synthetic polypeptides that mimic naturally occurringantigenic determinants on viral proteins. Data generated in studiesusing synthetic peptides have indicated that some of the conserveddomains in gp120 and gp41, respectively, contain immunodominant epitopesthat may be appropriate for diagnosis. From analysis of conserved HIVdomains from gp41 it appears that, within a linear sequence spanningabout 40 amino acids (amino acid 570-612), numerous and highlyimmunodominant epitopes of HIV reside. Wang, J. J. G., et al., Proc.Nat'l Acad. Sci. USA 83: 6159-63 (1986); Gnann, J. W., et al., J.Infect. Dis. 156: 261-67 (1987).

Conserved domains in gp120 and gp41 have also been identified that areinvolved in neutralization of different HIV isolates. Ho, D. D., et al.,J. Virol. 61: 2024-28 (1987); Science 239: 1021-23 (1988).Neutralizing-specific antibodies to the major envelope glycoprotein aretype-specific and have recently been mapped to a highly variablesequence of gp120. Putney, S. D., et al., J. Cell. Biochem. 12B: 5(1988). Neutralization assays with immune sera from animals or humans,see Robert-Guroff, M., et al., Nature 316: 72-74 (1985); Weiss, R. A.,et al., Nature 316: 69-71 (1985); Rasheed, S., et al., Virology 150: 1-6(1986), have revealed that the envelope proteins contains epitopes thatelicit antibodies capable of neutralizing HIV in vitro. But the presenceof these antibodies in vivo has only a limited effect on progression ofthe disease, Robert-Guroff, M., et al., loc. cit.; Wendler, I., et al.,AIDS Res. Human Retroviruses 3: 157-63 (1987), and no significantdifference in titers which can be correlated with clinical status. Inhumans, there are no known patterns of antibody response indicative ofimmunity.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a fusionprotein, readily produced in commercially significant quantities, thatis an effective vaccine for prevention of HIV infection.

It is a further object of the present invention to provide a fusionprotein, readily produced in commercially significant quantities, thatis an exceedingly sensitive and specific antigen for detection of HIV-1antibodies.

It is yet another object of the present invention to provide a highlyaccurate diagnostic assay for detecting antibodies to HIV-1.

In accomplishing these and other objects, there has been provided,according to one aspect of the present invention, a fusion proteincomprised of an amino acid sequence selected from the group consistingof NVTENFNMWKN, KAKRRVVQREKRAVG, ERYLKDQQLLGIWGCSGKLIC andEESQNQQEKNEQELLELDKWA; and a non-HIV polypeptide sequence, such that theamino-acid sequence and the polypeptide sequence comprise the backboneof the fusion protein, wherein the fusion protein reacts with anHIV-positive serum. In a preferred embodiment, the amino-acid sequenceis joined via a peptide bond to an N-terminus of the non-HIV polypeptidesequence.

In accordance with another aspect of the present invention, aself-replicating cell is provided that expresses a polypeptidecomprising an amino-acid sequence selected from the group consisting ofNVTENFNMWKN, KAKRRVVQREKRAVG, ERYLKDQQLLGIWGCSGKLIC andEESQNQQEKNEQELLELDKWA.

Also provided, according to still another aspect of the presentinvention, is a diagnostic assay for detecting the presence of anti-HIVantibody in a sample, comprising the steps of (A) immobilizing on asolid matrix a fusion protein comprising an amino-acid sequence,ERYLKDQQLLGIWGCSGKLIC and a non-HIV polypeptide sequence, such that theamino-acid sequence is accessible to an antibody contacting a surface ofthe matrix; (B) bringing a sample into contact with the surface of thematrix; and (C) monitoring the surface for binding of HIV-specificantibody. In one preferred embodiment, step (C) comprises detecing thepresence of anti-HIV antibody in a sample that tests HIV-negative whentested using a conventional whole-virus western blot assay.

Pursuant to another aspect of the present invention, a diagnostic assayis provided for detecting the presence of anti-HIV antibody in a sample,comprising the steps of (A) providing a fusion protein comprising anamino-acid sequence ERYLKDQQLLGIWGCSGKLIC and an amino acid sequence ofan enzyme; (B) combining a sample with the fusion protein in a liquid;and (C) monitoring the combination for a modulation of activity of theenzyme.

A vaccine comprising a fusion protein, as described above, and asterile, pharmacologically acceptable carrier therefor is also provided,in accordance with yet another aspect of the present invention.

In accordance with another aspect of the present invention, there hasbeen provided an immunotherapy method that comprises the step ofadministering to a subject an immunostimulatory amount of a vaccine asdescribed above. In a preferred embodiment, the subject is alreadyinfected with HIV-1 when the vaccine is administered.

Pursuant to another aspect of the present invention, an immunotherapymethod is provided comprising the step of administering to a subject animmunostimulatory amount of a hyperimmune globulin prepared according toa method comprised of immunizing a plasma donor with a vaccine asdescribed above, such that a hyperimmune globulin is produced whichcontains antibodies directed against HIV-1. According to another aspectof the present invention, an immunotherapy method is provided thatcomprises administering to a subject an immunostimulatory amount of ahyperimmune globulin prepared in the aforementioned manner.

Also provided is an immunotoxin conjugate comprising a fusion protein,as described above, conjugated to an immunotoxin. In addition, animmunotherapy method is provided, according to another aspect of thepresent invention, wherein a subject already infected with HIV-1receives antibodies directed against a fusion protein of the invention,wherein the antibodies are conjugated to an immunotoxin.

According to still another aspect of the present invention, acomposition is provided that consists essentially of antibodies thatbind the aforementioned fusion protein. (In this context, the qualifier“consists essentially of” means that the composition may have otherconstituents, such as a pharmaceutically acceptable carrier for theantibodies, but that the salient properties of the composition aredetermined by the immunological characteristics of the antibodies.) In apreferred embodiment, the composition is a monoclonal antibodycomposition, while in another preferred embodiment the antibodies arenot obtained by a process comprising the step of providing a biologicalsample from a human subject infected with HIV-1.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the oligonucleotide sequences coding for HIV88 (FIG. 1A),HIV500 (FIG. 1B), HIV582 (FIG. 1C) and HIV647 (FIG. 1D).

FIG. 2 shows the oligonucleotide sequences coding for SIV88 (FIG. 2A),SIV500 (FIG. 2B), SIV582 (FIG. 2C) and SIV647 (FIG. 2D). In both FIGS. 1and 2, the encoded amino acids for the respective polypeptides are alsoshown, as are the recognition sites for restriction endonucleases,denoted above each site, which can be used to obtain the oligonucleotidesequences from the HIV-1 genome.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

It has been discovered that certain short, highly conserved polypeptidesequences of gp41 and gp120, having from eleven to twenty-one aminoacids, possess attributes making them suitable for use in the diagnosis,treatment and prevention of HIV infection. According to the presentinvention, these polypeptide sequences are joined, preferably via DNArecombinant techniques, to a non-HIV protein or polypeptide sequencecomprising a sequence of amino acids not naturally found in the HIVgenome, to form a fusion protein.

Fusion proteins comprising these sequences have been shown to be goodcandidates for an HIV vaccine. While vaccines comprising these sequenceshave not been tested in humans, for obvious reasons, their utility inthis regard is indicated by results obtained when equivalent sequencesderived from SIVmac env are used to immunize Rhesus monkeys, discussedmore fully below. The HIV sequences according to the invention aredenominated HIV88, HIV500, HIV582 and HIV647, and have the followingamino acid sequences:

HIV88:

Asn-Val-Thr-Glu-Asn-Phe-Asn-Met-Trp-Lys-Asn (NVTENFNMWKN)

HIV500:

Lys-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg-Ala-Val-Gly(KAKRRVVQREKRAVG)

HIV582:

Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys(ERYLKDQQLLGIWGCSGKLIC)

HIV647:

Glu-Glu-Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala(EESQNQQEKNEQELLELDKWA)

One of these polypeptide sequences, HIV582, combines aspects of twopreviously delineated immunodominant epitopes of gp41(RILAVERYLKDQQLLGIWGCS and LGIWGCSGKLIC), and can be incorporated intofusion proteins that are consequently recognized by virtually allHIV-positive sera. Surprisingly, it can be used to detect the presenceof HIV-1-specific antibodies in sera that are obtained from patientsrecently infected by the virus and that test negative via whole-virusHIV-1 western blot and other conventional assays.

The identity of the non-HIV polypeptide to which the HIV polypeptidesequence is fused to form a fusion protein is not critical, but itpreferably is one that can be expressed by a genetically-engineeredmicrobe and purified from the culture medium. Exemplary of this group ofpolypeptides is β-galactosidase, protein G, acetylchloramphenicoltransferase, tryptophan synthetase, influenza A nonstructural protein(NS1), hepatitis core and surface antigens, and bacterial exotoxins suchas E. coli LT, cholera toxin, and Pseudomonas toxin A. Particularlypreferred are non-HIV polypeptides that possess an enzymatic propertythat can be exploited for purification and/or diagnostic purposes.Enzyme activity permits easier monitoring of purification. In addition,polypeptides according to the present invention in which the HIV582 isfused to an enzyme can form the basis for a simplified diagnostic assayusing a homogenous system in which modulation of enzyme activity ismonitored.

β-galactosidase is particularly preferred as the non-HIV polypeptidefused to the HIV polypeptide sequence for other reasons. In addition tothe advantages arising the fact that it is an enzyme, it possesses otherbeneficial attributes. Since it is a tetramer it can hold four epitopes.Another significant advantage, and one that is entirely unexpected, isthe fact that most people do not have any antibodies to β-galactosidase.

When an HIV polypeptide sequence is fused to β-galactosidase, theβ-galactosidase acts as an immunocarrier, i.e., a substance, usually apolypeptide or protein, which is critical for the efficient interactionbetween T and B cells for the induction of an immune response against asmall antigen that is attached to it.

A fusion protein within the present invention incorporates the HIVpolypeptide sequence into the primary structure (“backbone”) of theprotein. It is preferred that the HIV polypeptide sequence be joined,via a peptide bond between the terminal carboxyl group of the HIVsequence and the terminal amino group of the non-HIV polypeptide. Thisarrangement enhances the possibility of a reaction between the fusionprotein and antibodies that develop as a consequence of HIV-1 infection.Moreover, the arrangement is efficient in inducing an immune responsethat is specific for HIV-1.

The fusion proteins of the present invention can be produced byconventional genetic-engineering techniques. In this regard, apolynucleotide molecule encoding the desired protein preferablycomprises a nucleotide sequence, corresponding to one of the amino-acidsequences according to the invention, that is optimized for the host ofchoice, such as E. coli, in terms of codon usage and initiation oftranslation. In the same vein, the vector selected for transforming thehost organism with the polynucleotide molecule should allow forefficient maintenance and transcription of the sequence coding for thefusion protein.

Fusion proteins comprising HIV582 are particularly useful in variousimmunoassays which detect the presence of antibodies indicative of aparticular disease state. For example, western blot and ELISA immobilizethe polypeptides on a solid matrix and then contact the immobilizedpolypeptide with a sample.

The acronym ELISA refers to “enzyme-linked immunosorbent assay,” thatis, an assay using an antigen or antibody bound to a solid phase and anenzyme-antibody or enzyme-antigen conjugate to detect and/or quantifyantibody or antigen present in a sample. In western blot assays,solubilized and separated antigens are bound to nitrocellulose paper.The antibody is detected by an enzyme or label-conjugatedanti-immunoglobulin (Ig), such as horseradish peroxidase-Ig conjugateand detected by incubating the filter paper in the presence of aprecipitable or detectable substrate. Western blot assays have theadvantage of not requiring purity greater than 50% for the desiredpolypeptide. Descriptions of the ELISA and western blot techniques arefound in Chapters 10 and 11 of Ausubel, et al. (eds.), CURRENT PROTOCOLSIN MOLECULAR BIOLOGY, John Wiley and Sons (1988), the contents of whichare hereby incorporated by reference.

The present invention also relates to the use of a fusion protein toproduce antisera or monoclonal antibodies (mouse or human) that bind toor neutralize virus. These antibodies can be employed to produceimmunotoxin conjugates (e.g., with a ricin A chain) which should beuseful in therapy of HIV-1 infections. Protocols for producing theseantibodies are described in Ausubel, et al. (eds.) loc. cit., Chapter11; in METHODS OF HYBRIDOMA FORMATION 257-71, Bartal & Hirshaut (eds.),Humana Press, Clifton, N.J. (1988); in Vitetta et al., Immunol. Rev. 62:159-83 (1982); and in Raso, Immunol. Rev. 62: 93-117 (1982).

Inocula for polyclonal antibody production are typically prepared bydispersing the dried solid HIV sequence-immunocarrier in aphysiologically tolerable diluent such as saline to form an aqueouscomposition. An immunostimulatory amount of inoculum is administered toa mammal and the inoculated mammal is then maintained for a time periodsufficient for the HIV sequence to induce protecting anti-HIV-1antibodies.

Antibodies can include antiserum preparations from a variety of commonlyused animals, e.g., goats, primates, donkeys, swine, rabbits, horses,hens, guinea pigs, rats or mice, and even human antisera afterappropriate selection and purification. The animal antisera are raisedby inoculating the animals with an immunogenic form of the pathogen orits antigen, by conventional methods, bleeding the animals andrecovering serum or an immunoglobulin-containing serum fraction.

The antibodies induced in this fashion can be harvested and isolated tothe extent desired by well known techniques, such as by immunoaffinitychromatography; that is, by binding antigen to a chromatographic columnpacking like Sephadex®, passing the antiserum through the column,thereby retaining specific antibodies and separating out otherimmunoglobulins and contaminants, and then recovering purifiedantibodies by elution with a chaotropic agent, optionally followed byfurther purification, for example, by passage through a column of boundblood group antigens or other non-pathogen species. This procedure maybe preferred when isolating the desired antibodies from the serum ofpatients having developed an antibody titer against the pathogen inquestion, thus assuring the retention of antibodies that are capable ofbinding to exposed epitopes. They can then be used in preparations forpassive immunization against HIV-1 or in diagnostic assays.

A monoclonal antibody composition contains, within detectable limits,only one species of antibody combining site capable of effectivelybinding HIV-1 TMP. In particular, a monoclonal antibody composition ofthe present invention typically displays a single binding affinity forHIV-1 TMP even though it may contain antibodies capable of bindingproteins other than HIV-1 TMP.

Suitable antibodies in monoclonal form can be prepared usingconventional hybridoma technology. To form a hybridoma from which amonoclonal antibody composition of the present invention is produced, amyeloma or other self-perpetuating cell line is fused with lymphocytesobtained from peripheral blood, lymph nodes or the spleen of a mammalhyperimmunized with a polypeptide of this invention. It is preferredthat the myeloma cell line be from the same species as the lymphocytes.Splenocytes are typically fused with myeloma cells using polyethyleneglycol 1500. Fused hybrids are selected by their sensitivity to HAT.Hybridomas secreting the antibody molecules of this invention can beidentified using an ELISA.

A Balb/C mouse spleen, human peripheral blood, lymph nodes orsplenocytes are the preferred materials for use in preparing murine orhuman hybridomas. Suitable mouse myelomas for use in the presentinvention include the hypoxanthine-aminopterin-thymidine-sensitive (HAT)cell lines P3X63-Ag8.653 and Sp2/0-Ag14, available from the AmericanType Culture Collection, Rockville, Md., under the designations CRL 1580and CRL 1581, respectively. The preferred fusion partner for humanmonoclonal antibody production is SHM-D33, a heteromyeloma availablefrom ATCC, Rockville, Md. under the designation CRL 1668.

A monoclonal antibody composition of the present invention can beproduced by initiating a monoclonal hybridoma culture comprising anutrient medium containing a hybridoma that secretes antibody moleculesof the appropriate polypeptide specificity. The culture is maintainedunder conditions and for a time period sufficient for the hydridoma tosecrete the antibody molecules into the medium. The antibody-containingmedium is then collected. The antibody molecules can then be furtherisolated by well known techniques.

Media useful for the preparation of these compositions are both wellknown in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's Minimal essential medium supplemented with 4.5 g/lglucose, 20 mm glutamine, and 20% fetal calf serum. An exemplary inbredmouse strain is the Balb/c.

Other methods of preparing monoclonal antibody compositions are alsocontemplated, such as interspecies fusions and genetic engineeringmanipulations of hypervariable regions, since it is primarily theantigen specificity of the antibodies that affects their utility in thepresent invention. Human lymphocytes obtained from HIV-infectedindividuals can be fused with a human myeloma cell line to producehybridomas which can be screened for the production of antibodies thatrecognize a fusion protein of the present invention. More preferable inthis regard, however, is a process that does not entail the use of abiological sample from a human subject infected with HIV-1. For example,a subject immunized with a vaccine as described herein can serve as asource for antibodies suitably used in an antibody composition withinthe present invention.

The monoclonal and polyclonal antibody compositions produced accordingto the present description can be used to induce an immune response forthe prevention or treatment of HIV infection. They can also be used indiagnostic assays where formation of an HIV-1 TMP-containingimmunoreaction product is desired.

In this regard, the antibody component can be polyspecific, that is, itcan include a plurality of antibodies that bind to a plurality ofepitopes represented by the various conserved polypeptide sequencesdescribed above. The polyspecific antibody component can be a polyclonalantiserum, preferably affinity purified, from an animal which has beenchallenged with a fusion protein of the present invention and, hence,stimulated to produce a plurality of specific antibodies against thefusion protein. Another alternative is to use an “engineered polyclonal”mixture, which is a mixture of monoclonal antibodies with a definedrange of epitopic specificities.

In both types of polyclonal mixtures, it can be advantageous to linkpolyspecific antibodies together chemically to form a singlepolyspecific molecule capable of binding to any of several epitopes. Oneway of effecting such a linkage is to make bivalent F(ab′)₂ hybridfragments by mixing two different F(ab′)₂ fragments produced, e.g., bypepsin digestion of two different antibodies, reductive cleavage to forma mixture of Fab′ fragments, followed by oxidative reformation of thedisulfide linkages to produce a mixture of F(ab′)₂ fragments includinghybrid fragments containing a Fab′ portion specific to each of theoriginal antigens. Methods of preparing such hybrid antibody fragmentsare disclosed in Feteanu, LABELED ANTIBODIES IN BIOLOGY AND MEDICINE321-23, McGraw-Hill Int'l Book Co. (1978); Nisonoff, et al., ArchBiochem. Biophys. 93: 470 (1961); and Hammerling, et al., J. Exp. Med.128: 1461 (1968); and in U.S. Pat. No. 4,331,647.

Other methods are known in the art to make bivalent fragments that areentirely heterospecific, e.g., use of bifunctional linkers to joincleaved fragments. Recombinant molecules are known that incorporate thelight and heavy chains of an antibody, e.g., according to the method ofBoss et al., U.S. Pat. No. 4,816,397. Analogous methods of producingrecombinant or synthetic binding molecules having the characteristics ofantibodies are included in the present invention. More than twodifferent monospecific antibodies or antibody fragments can be linkedusing various linkers known in the art.

An antibody component produced in accordance with the present inventioncan include whole antibodies, antibody fragments, or subfragments.Antibodies can be whole immunoglobulin (IgG) of any class, e.g., IgG,IgM, IgA, IgD, IgE, chimeric antibodies or hybrid antibodies with dualor multiple antigen or epitope specificities, or fragments, e.g.,F(ab′)₂, Fab′, Fab and the like, including hybrid fragments, andadditionally includes any immunoglobulin or any natural, synthetic orgenetically engineered protein that acts like an antibody by binding toa specific antigen to form a complex. In particular, Fab molecules canbe expressed and assembled in a genetically transformed host like E.coli. A lambda vector system is available thus to express a populationof Fab's with a potential diversity equal to or exceeding that ofsubject generating the predecessor antibody. See Huse, W. D., et al.,Science 246: 1275-81 (1989), the contents of which are herebyincorporated by reference.

A polypeptide according to the present invention can be the activeingredient in a composition, further comprising a pharmaceuticallyacceptable carrier for the active ingredient, which can be used as avaccine to induce a cellular immune response and/or production in vivoof antibodies which combat HIV-1 infection. In this regard, apharmaceutically acceptable carrier is a material that can be used as avehicle for administering a medicament because the material is inert orotherwise medically acceptable, as well as compatible with thepolypeptide active agent, in the context of vaccine administration. Inaddition to a suitable excipient, a pharmaceutically acceptable carriercan contain conventional vaccine additives like diluents, adjuvants,antioxidants, preservatives and solubilizing agents.

Pursuant to the present invention, such a vaccine can be administered toa subject not already infected with the virus, thereby to induce anHIV-protective immune response (humoral or cellular) in that subject.Alternatively, a vaccine within the present invention can beadministered to a subject in which HIV-1 infection has already occurredbut is at a sufficiently early stage that anti-HIV antibodies producedin response to the vaccine effectively inhibit further spread ofinfection.

By another approach, a vaccine of the present invention can beadministered to a subject who then acts as a source for globulin,produced in response to challenge from the specific vaccine(“hyperimmune globulin”), that contains antibodies directed againstHIV-1. A subject thus treated would donate plasma from which hyperimmuneglobulin would then be obtained, via conventional plasma-fractionationmethodology, and administered to another subject in order to impartresistance against or to treat HIV-1 infection. Similarly, monoclonal orpolyclonal anti-HIV-1 antibodies produced using a fusion proteinaccording to the present invention can be conjugated to an immunotoxin,as described above, and administered to a subject in whom HIV-1infection has already occurred but has not become widely spread. To thisend, antibody material produced pursuant to the present descriptionwould be administered in a pharmaceutically acceptable carrier, asdefined herein.

The present invention is further described below by reference to thefollowing, illustrative examples. In keeping with standard polypeptidenomenclature, the following abbreviations shown below for amino acidresidues are used.

TABLE OF CORRESPONDENCE Ala A Alanine Leu L Leucine Arg R Arginine Lys KLysine Asn N Asparagine Met M Methionine Asp D Aspartic acid Phe FPhenylalanine Cys C Cysteine Pro P Proline Glu E Glutamic Acid Ser SSerine Gln Q Glutamine Thr T Threonine Gly G Glycine Trp W TryptophanHis H Histidine Tyr Y Tyrosine Ile I Isoleucine Val V Valine

In order to test the level of antibody production elicited by theadministration of a polypeptide of the present invention, two differentprotocols were used. In the first, Rhesus monkeys were immunized with anHIV sequence fused to β-galactosidase, and the antibody titer wasmeasured at various times after immunization.

In the second technique, sequences were identified from SIVmac (SimianImmunodeficiency Virus) env that are equivalent, according to thefollowing criteria, to the sequences set forth in Table 1. SIV peptidesequences on SIVmac env that appeared in a location similar to thatfound in the primary sequence of HIV-1 env, which were also hydrophilicand at least about 50% homologous to the sequence to the HIV-1 envpeptide, were selected.

Of the six epitopes shown in Table 1 below, only three SIVmac envsequences, SIV88, SIV582 and SIV647, satisfied all three criteria. Afourth SIV sequence, SIV500, which showed only about 25% homology toHIV500, was also selected, because this epitope was found to be animmunodominant C-terminal epitope on HIV-1 gp120. SIV500 corresponds tothe putative C-terminal SIV gp120 sequence. Like HIV500, it washydrophilic.

Because of the criteria used to correlate HIV and SIV sequences,observation of the antibody response of monkeys immunized with an SIVsequence could be used to assess the antibody response of humansimmunized with the corresponding HIV sequence. The four SIV polypeptidesequences selected are shown below.

SIV88:

Asn-Val-Thr-Glu-Ser-Phe-Asp-Ala-Trp-Glu-Asn (NVTESFDAWEN)

SIV500:

Arg-Tyr-Thr-Thr-Gly-Gly-Thr-Ser-Arg-Asn-Lys-Arg (RYTTGGTSRNKR)

SIV582:

Glu-Lys-Tyr-Leu-Glu-Asp-Gln-Ala-Gln-Leu-Asn-Ala-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys(EKYLEDQAQLNAWGCAFRQVC)

SIV647:

Glu-Glu-Ala-Gln-Ile-Gln-Gln-Glu-Lys-Asn-Met-Tyr-Glu-Leu-Gln-Lys-Leu-Asn-Ser-Trp-Asp(EEAQIQQEKNMYELQKLNSWD)

EXAMPLE 1 Production and Purification of Recombinant Fusion Protein

From published amino-acid sequences, hydrophobicity plots of the HIV envencoded proteins were generated, and five domains were selected, eachmanifesting conservation of sequences and a high degree ofhydrophilicity, and a high content of amino acids implicated in thedetermination of the secondary structure on a protein, such as cysteine,aromatic amino acids, proline and glycine. Sequences rich in hydrophobicresidues were avoided, since highly hydrophilic regions are more likelyto be antigenic than are hydrophobic regions. Three peptides, designatedHIV88, HIV475 and HIV500 in Table 1 below, were selected from theNH₂-terminus and COOH-terminus of gp120, and two peptides from theNH₂-terminus portion of gp41 (HIV647 and HIV705 in Table 1). Alsoprepared was a sixth domain (HIV582 in Table 1) from gp41, combiningaspects of two highly immunodominant sequences. See Wang, J. J. G., etal., loc. cit.; Gnann, J. W., et al., loc. cit.

Oligonucleotides coding for the HIV amino-acid sequences depicted inTable 1 were synthesized on a 381A DNA synthesizer (Applied Biosystems,Foster City, Calif.). The sequences of these oligonucleotides are shownin FIG. 1. The oligonucleotides were purified and inserted into plasmidpTZ₂ or a derivative thereof, pTOZ or pTIZ, pursuant to methodology ofShaffermann, A., et al., J. Biol. Chem. 262: 6227-37 (1987), thecontents of which are hereby incorporated by reference. These plasmidswere designed to accommodate various synthetic oligonucleotides in framewith the NH₂-terminus of β-galactosidase coding sequences, and to beexpressed as fusion polypeptide under control of the E. coli trppromoter. See Ausubel, et al. (eds.), loc. cit., chapter 13, thecontents of which are hereby incorporated by reference.

The synthetic oligonucleotides coding for HIV88 and HIV500 were clonedbetween the HindIII and PstI sites of pTOZ; HIV582, HIV647 and HIV705between the NcoI and HindIII sites of pTOZ and HIV475 between the NdeIand HIndIII sites of pTZ₂. Correct constructs were verified byrestriction enzyme analysis and DNA sequencing.

Strains of E. coli MC1060, see Casadaban, M. J., et al.,“β-galactosidase gene fusions for analyzing gene expression in E. coliand yeast,” in 100 METHODS OF ENZYMOLOGY 293-308 (Academic Press, NewYork), were transformed with the various HIV-pTOZ, HIV-PTIZ or HIV-pTZ₂plasmids. The recombinant E. coli bacteria were maintained by serialpassage on Lauria broth agar plates containing 50 μg/ml of ampicillin.

The E. coli were induced to express β-galactosidase under conditionsdescribed by Grosfeld, H., et al., loc. cit., and Shaffermann, A., etal., loc. cit. For scale up, an isolate was serially diluted in Lauriabroth and ampicillin and allowed to grow overnight at 37 C withagitation. An inoculum from the overnight culture was transferred to anutritionally deficient, defined medium (M9 media with limitedL-tryptophane) and shaken vigorously at 37 C until an optical density of3.0 (A₅₅₀) was achieved.

The cultured bacteria were centrifuged and the supernatant wasdiscarded. The resulting pellet was treated with lysozyme for 30minutes, and lysed by serial freezing and thawing. The lysate wascentrifuged at high speed and the supernatant was recovered.

Recombinant protein of the present invention was precipitated from thelysate using ammonium sulfate. The level of production for each HIV-galwas in range of 2% to 10% of total bacterial proteins. Optimal recoverywas found in the 40% cut. The precipitate was resuspended in a Trisbuffer containing 2-mercaptoethanol, and dialyzed to remove excesssalts. The resulting solution contained approximately 50% purerecombinant protein, mixed with other E. coli proteins of nonrecombinantorigin, as characterized by Coomasie blue stained SDS-PAGE gels.

In a conventional manner, recombinant protein of the present inventioncan be further purified by affinity chromatography, as necessary, by useof a gel matrix upon which is immobilized, as a ligand, monoclonalantibody against beta-galactosidase. Alternatively, the fusion proteincan be purified by high-performance liquid chromatographic ion exchange(DEAE5PW Waters Column), yielding over 98% pure protein. Either methodwould yield fusion protein with greater than 95% purity.

SIV recombinant fusion proteins were made in a similar manner, witholigonucleotides coding for the equivalent SIV amino-acid sequencesbeing used. These oligonucleotides are shown in FIG. 2. Sequences of theSIV envelope are according to those published for SIVmac. See Franchini,G., et al., Nature 328: 539-42 (1987).

EXAMPLE 2 Evaluation of Reactive Specificity for Selected Domains of HIVEnvelope Protein

As a first step in evaluation of the selected HIV env conserved domains,the reactivity of the six different HIV-gal fusion polypeptides wasanalyzed with specific panels of HIV-seropositive sera collected fromvarious stages of HIV infection.

Serum samples were collected from patients having two separate serumspecimens positive for anti-HIV antibodies by ELISA and western blot.See Redfield, R. R., et al., New Engl. J. Med. 314: 131-32 (1986).Virus-positive samples were defined as those in which a simultaneousculture of the patient's peripheral blood monocytes (PBMCs) yielded apositive culture by either RT activity or antigen capture, according toGallo, D., et al., J. Clin. Microb. 25: 1291-94 (1987).

To assay the sera, an equivalent of 15 ng of HIV peptides (0.7-1.5 μg of98% pure HIV-β-galactosidase) were dotted in duplicates onnitrocellulose filter disks. Filters were blocked with 1% casein/BSA for2 hours at room temperature and then incubated for 18 hours at roomtemperature with four fold serial dilutions of sera, starting from adilution of 1:100. Filters were washed and incubated with goatanti-(IgG, IgM) human antibodies conjugated to phosphatase (KirkegaardPerry) at a final concentration of 3 ng/ml and developed (with Fast redTR salt and naphthol AS-MX phosphate Sigma) for 30 minutes. Each filterwas dotted with all the HIV-βgal proteins as well as with a control ofβ-galactosidase. The end point of titration was determined by threeindividuals. Out of a total of 1360 readings of HIV-gal titrations,complete accordance was achieved in 1350 cases.

As shown in Table 1, each of the HIV-derived peptides were recognized byantibodies elicited in some or all the HIV-infected individuals. None ofthe twenty control sera reacted with the HIV-βgal polypeptides.

TABLE 1 Amino Acid Sequence of Various HIV-β-Galactosidase FusionProteins and Their Reactivities with HIV Seropositive Sera. HIV-AminoAcid Number Positive/Total HIV-gal Sequence (percent positive) HIV88NVTENFNMWKN 32/75 (43) HIV475 MRDNWRSELYKYKV  8/30 (27) HIV500KAKRRVVQREKRAVG 36/75 (48) HIV582 ERYLKDQQLLGIWGCSGKLIC  75/75 (100)HIV647 EESQNQQEKNEQELLELDKWA 25/30 (83) HIV705 VNRVRQGYSPLSFQT  5/30(17)

In general, a decrease in titers against the HIV domains testedheretofore has been observed with progression of disease. It issignificant as well as unexpected, therefore, that all of theHIV-positive sera not only reacted with HIV582, but also responded intiters (10⁻⁵) that were almost two orders of magnitude higher than anyof the other conserved envelope regions (see Table 2). By contrast,HIV500, which overlaps most of the sequences of SP22 previouslyidentified as a major immunodominant epitope on gp120 by Parker, T. J.,et al., loc. cit., was recognized in only 48% of HIV positive sera andonly elicited an antibody titer in the range of 1:1500 in theHIV-infected individual.

The increased magnitude of response is a result of the high level ofexpression of HIV582 at all stages of infection as compared to otherepitopes. This difference in level of expression vis-a-vis otherepitopes is especially high during early stages of infection, allowingHIV582 to successfully detect infection at a much earlier stage thatother epitopes. The HIV582 sequence is thus shown to provide a sensitivetool for diagnostic purposes, capable of detecting very early stages ofinfection.

TABLE 2 Differential Reactivities of HIV-βgal Polypeptides with Serafrom WR Stages 1 & 2 Versus WR Stages 5 & 6. Positive/Total GeometricMean Titer (%) of Positive Specimens HIV-βgal WR 1 & 2 WR 5 & 6 WR 1 & 2WR 5 & 6 HIV88  29/47* 3/28* 1:780 1:1,260 (62) (11) HIV475 6/17 2/131:710 1:200 (35) (15) HIV500 25/47 11/28 1:1,450 1:1,000 (53) (39)HIV582 47/47 28/28 1:140,000 1:78,000 (100) (100) HIV647 14/17 11/131:3,600 1:1,550 (82) (85) HIV705 4/17 1/13 1:1,000 1:600 (23) (8)*Difference in proportion positive is statistically significant (p <0.00001 by Fisher Exact test).

Of the two other conserved epitopes in gp41 that have been studied, theone within HIV647 also seems to be immunodominant. HIV647-βgal wasrecognized by 83% of HIV positive tested sera in any stage of thedisease and in relatively high titers, as shown in Table 2. On the otherhand, no reaction with HIV-positive sera was observed when a verysimilar peptide, CQNQQEKNEQELLE, was used. Gnann, J. W., et al., loc.cit. This may reflect the presence of the extra amino acids in HIV647,or it may result from the higher sensitivity of the immunodot methodused. Alternatively, it may be that the coupling of this peptide tokeyhole limpet hemocyanin results in conformational changes notoccurring in HIV647-βgal.

Further study was made of an additional 45 sera from patients whosePBMCs were cultured for virus isolation, following Gallo, D., et al., J.Clin. Microbiol. 25: 1291-94 (1987). Within this group studied, twentypatients were virus isolation-negative (all in Walter Reed stages 1 or2) and twenty-five patients were virus isolation-positive (ten in WRstages 1 or 2, and fifteen in WR stages 5 or 6). Since success of virusisolation is only 10-15% from PBMC specimens from patients in earlystage disease, but close to 100% from patients in late stage disease,the sera selected for testing were intentionally biased in favor ofvirus culture-positive, early-stage patients.

As shown in Table 3, thirteen of the twenty HIV culture-negativepatients (65%) had detectable HIV88-βgal reactivity, while only five oftwenty-five sera from HIV isolation-positive patients had detectableHIV88-βgal antibodies (p<0.003). Among sera from early-stage patients(WR 1 or 2) only, thirteen of twenty specimens from virusculture-negative patients had HIV88-βgal reactivity, compared to onlythree of ten virus culture-positive patients (p=0.077). Antibodies toHIV500-βgal and HIV582-βgal were detected equally often in sera fromvirus isolation-positive and -negative patients. But unlike HIV500,HIV582 was positive in every tested case, regardless of the stage of thedisease or the ability to detect virus in PBMC culture.

TABLE 3 Distribution of Reactivities of HIV-βgal Polypeptides withSelected Sera from Patients with Positive or Negative Virus Isolationfrom PBMCs. Patient Sera Stage 1-2 Stage 1-2 Stage 5-6 HIV-βgal VirusNeg Virus Pos Virus Pos HIV-88  13/20 3/10 2/15 HIV-500  9/20 6/10 4/15HIV-582 20/20 10/10  15/15 

EXAMPLE 3 Antigenicity of the Recombinant Protein Versus Known SyntheticPeptide Conjugated to Bovine Serum Albumin

By means of a dot immunoassay, the antigenicity of the fusion proteinwas found to be far superior to a commercially synthesized peptide(Peninsula, Belmont, Calif.) which consisted of the same twenty-oneamino acids of HIV582 conjugated to bovine serum albumin (BSA). Thispeptide conjugate was prepared such that BSA and the twenty-one aminoacid sequence of HIV582 were present in equivalent weights, i.e., one mgof conjugate contained 500 ng of HIV582 and 500 ng of BSA. Ammoniumsulfate-precipitated recombinant HIV antigen was used at 50% purity, andthe weight of the HIV582 was calculated to be 10 ng/mg total protein inthe antigen solution. The recombinant and peptide conjugate antigenswere prepared as 1 mg/ml total protein solutions (determined by theLowry method) and 5 μl samples of serially diluted antigen were appliedto nitrocellulose paper, air dried, and blocked. It was confirmed thatequivalent amounts of total protein were bound to the nitrocellulose byreaction with ninhydrin. The paper was incubated for four hours withserum containing HIV antibodies diluted 1:100, and antigen-boundantibody was detected with a peroxidase-conjugated secondary antibodysystem. The results are presented in Table 4, where the intensity of thevisible dot was graded on a scale in which ± was a barely detectablereaction, and ++++ was an intensely dark dot on the nitrocellulosepaper. These results indicate that the recombinant HIV582 antigen is atleast 50 times more sensitive than the synthetic peptide conjugate thatwas used in the diagnostic assay.

TABLE 4 Comparison of Recombinant HIV582-βgal and Synthetic HIV582-BSAConjugate. Recombinant HIV582-βgal Synthetic HIV582-BSA Antigen (ng)Reactivity Antigen (ng) Reactivity 50 ++++ 2500 ++++ 25 ++++ 1250 ++++12.5 ++++ 625 +++ 6.25 ++++ 312.5 +++ 3.13 +++ 156.25 ++ 1.57 +++ 78.12++ 0.78 ++ 39.10 + 0.39 + 19.55 +/−

EXAMPLE 4 Comparative Study of Fusion Protein-Based Western Blot andConventional Whole-Virus HIV-1 Western Blot

Western blot strips containing the fusion protein were prepared bySDS-PAGE followed by transfer to nitrocellulose paper. Strips containingapproximately 2 μg of fusion protein were cut from the nitrocellulosestrips and used to characterize a pool of 400 sera from HIV-infectedpatients as determined from clinical symptoms, whole-virus western blotor virus isolation, and 500 HIV-negative sera at 1:100 dilution. All 400HIV-positive samples reacted with the fusion protein, while none of the500 negative samples reacted with the antigen. The specificity andsensitivity of the test with this sample population were thus 100%.

A subpopulation of 104 sera from the pool of 400 was characterized usinga the conventional whole-virus HIV-1 western blot (DuPont). According tothe manufacturer, the presence of antibodies to three specific antigens,gag, env and RT, are required for a positive diagnosis. Many physicians,however, positively diagnose the disease based on the presence ofantibodies to only two antigens, env and either gag or RT. The resultswith the whole-virus western blot are shown in Table 5.

TABLE 5 Sera Reactivity with Whole-virus Western Blot. Antigen Number ofSera gag, env and RT 84 env and gag or 11 env and RT env only 4 gag only4 no reaction 1

Thus, even under the more relaxed “two out of three” criteria, theconventional whole-virus western blot gives a false negative in over 8%of the samples. Most importantly, the whole virus failed to identify oneof the sera as positive under any criteria. This individual was lateridentified as HIV positive when results of his virus isolation werecomplete.

The diagnostic assay based on HIV582 correctly identified all 104infected individuals. Thus, the HIV582 assay identifies infectedindividuals that do not give a positive reaction with any of the threeantigens used in whole virus western blot assay, allowing HIV positiveindividuals to be identified at an earlier stage of infection.

EXAMPLE 5 Antibody Titers to Various SIVmac env Peptides in Plasma fromDifferent Macaque Species Infected with SIVMne

Three juvenile Rhesus macaques (M. mulatta), three juvenile pigtailedmacaques (M. nemestrina), and two cynomolgus macaques (M. fascicularis)were inoculated intravenously with 10³ TCID of SIV/Mne. See Shaffermann,A., et al., J. Aids Res. 5: 327-26 (1989), the contents of which areincorporated herein by reference. All macaques became viremic withinthree weeks after inoculation, and all mounted an antibody response toSIV/Mne except M. nemestrina T85056, which died at fifteen weeks with anulcerative necrotizing colitis and a marked decrease in CD4+ PBL. Theother macaques died 43-121 weeks after inoculation after exhibitingprogressive weight loss, anemia, and diarrhea. Histopathologic findingsat necropsy included various manifestations of immune deficiency.

The development of antibodies of SIV/Mne was determined by esternimmunoblotting at various times after inoculation, and is tabulated inTable 5. Seven macaques developed readily detectable antibodies by 5-6weeks after inoculation, with antibodies to gag p28 and to thetransmembrane protein p34E generally appearing before antibodies toother gene products was detected. At various times after inoculation,antibodies to gp120 (the envelope protein) and to gag proteins p16, p8and p6 were also evident. In addition, some of the macaques developedantibodies to p14, which has been identified as the product of the X-orfgene of SIV.

Prior to inoculation of monkeys with SIV/Mne, none of the primate sera(dilution 1:100) reacted with the SIVmac env-gal polypeptides, yet allSIVmac env-βgal polypeptides reacted with antibodies elicited in all thepost-SIV/Mne infected monkeys. Pig-tailed macaque T85056, althoughviremic, remained antibody-negative after inoculation. Plasma from thisanimal did not react with the SIVmac env-βgal polypeptides. The order ofboth antibody prevalence to the various SIV env epitopes and theimmunogencity of these epitopes in SIV-infected macaques isSIV-582>SIV-647>SIV-500>SIV-88. The order of antibody prevalence and ofimmunogenicity of these envelope epitopes is identical to that found forthe equivalent HIV envelope epitopes in humans infected with HIV-1.

The results show that the humoral response in macaques infected withSIVMne to the specific SIVmac envelope epitopes parallels that observedfor the equivalent HIV env epitopes in humans infected with HIV. Thisshows that the SIV macaque system is a suitable model for assessing HIVvaccines and other immunotherapies for AIDS.

TABLE 5 Antibody response to SIVmac env peptides from macaques infectedwith SIVMne. Weeks after Antibody titer Animal infection SIV88 SIV500SIV582 SIV647 M. mulatta A85033 9 <1:100 <1:100 1:6400 <1:100 36 <1:100 1:200 1:100K <1:100 66 <1:100 <1:100 1:100K  1:400 A85034 14 <1:100<1:100 1:25K <1:200 36 <1:100 <1:100 1:50K  1:1600 87 <1:100 <1:1001:200K  1:6400 A85037 14 <1:100  1:400 1:1600 <1:100 36 <1:100  1:16001:25K <1:100 95 <1:100  1:1600 1:6400  1:100 M. nemestrina F85062 18<1:100  1:400 1:100K  1:600 36  1:400  1:400 1:400K  1:1600 80 <1:100 1:400 1:100K  1:1600 M85026 18  1:400 <1:100 1:25K  1:6400 66  1:400<1:100 1:100K  1:100K 120  1:200 <1:100 1:100K  1:400K M. fascicularis85175 12  1:100  1:100 1:6400  1:100 37  1:100  1:100 1:12,500 <1:100 51 1:400 <1:100 1:50K <1:100 85176 12  1:200  1:1600 1:25K  1:200 24 1:400  1:3200 1:100K  1:400 43  1:200  1:6400 1:100K  1:400 *Plasmafrom all macaques were seronegative for all SIV env-gal polypeptides ata dilution of 1:100 prior to inoculation with SIVMne.

EXAMPLE 6 Immunization of M. mulatta with HIV647-βgal

Three groups of monkeys (3 animals/group) were immunized with differentdoses of HIV647-βgal: 4, 40 and 400 μg (95% HPLC pure). On day zero,each animal received complete Freund's, on day 14 each animal receivedincomplete Freund's, and on day 35 each animal received soluble antigenonly. Adjuvant was mixed (0.5 ml with 0.5 ml HIV-βgal prior toinoculation. Monkeys were inoculated intra-muscularly at four sites with0.25 ml per site.

The antibody response was measured by an immunodot test in which 1.0 μgof 98% pure antigen (HIV647-βgal, HIV88-βgal and βgal) was dotted induplicate on nitrocellulose filters. The filters were blocked with 1%casein/BSA for two hours. Diluted sera were preincubated with βgal, atconcentrations of 200−80 μg/ml, for one hour at 37° C. and thenincubated for eighteen hours at room temperature with the filters.Filters were washed, then incubated with goat anti-human phosphataseantibodies (3 ng/ml) for three hours and developed for nine minutes.

An ELISA was also performed with a synthetic twenty amino acid peptideof the HIV647 epitope. ELISA was performed on PVC plates preincubatedwith 4 μg/ml of a cys-647 HIV peptide, a synthetic peptide of HIV647 towhich a cysteine residue was added at the N-terminus. Linearity of ODreadings with sera dilution was not observed. Differential (Day x−Dayzero) OD values for each dilution were used to determine the endpoint. Asignificant differential OD value was considered to be that which wasthreefold higher than that obtained from the differential value of Day14−Day Zero and above a cutoff value of 0.040. No attempt was made tooptimize the sensitivity of the ELISA.

On day zero, no sera had detectable antibody to HIV647 at a serumdilution of 1:100 with immunodot or 1:8 with ELISA. On day 14, all serahad titers lower than 1:1000. None of the sera reacted with a control ofHIV88-βgal. The antibody titers on days 28 and 51 as measured by bothimmunodot and ELISA are shown in Table 7.

TABLE 6 Antibody response to HIV647-βgal measured by immunodots andELISA. Immunodot Titer ELISA titer Dose Animal Day 28 Day 51 Day 28 Day51 4 T313 1:2000 1:64K <1:8 1:256 4 O27A 1:16K  1:64K   1:512  1:1024 4T324 <1:1000  1:32K <1:8  1:1024 40 O5 1:4000 1:32K <1:8 <1:8   40 X6621:16K  1:64K  1:64 1:128 40 T308 n.m. n.m.   1:128 1:128 400 T34 1:16K 1:32K  1:32 1:128 400 P792 1:8000 1:16K  1:16 1:128 400 A32 1:2000 1:64K<1:8 1:128 n.m. - not measurable due to βgal background

The fact that the sera reacted with HIV647-βgal, but not withHIV88-βgal, indicates that the response is specific to HIV647. The ELISAresults confirm that, while HIV647-βgal is composed of both HIV647 andβgal, antibodies are produced against the HIV647 sequence.

The antibody response to the βgal immunocarrier was also measured byimmunodot titration. All sera on day zero had anti-βgal titers lowerthan 1:50. The results on days 14, 28 and 51 are shown in Table 7.

TABLE 7 Antibody response to the βgal immunocarrier. Immunodot TiterDose Animal Day 14 Day 28 Day 51 4 T313 <1:500 1:50K 1:250K 4 O27A<1:500 1:50K 500K 4 T324 <1:500 1:500 1:25K 40 O5  1:5000 1:50K 1:50K 40X662  1:5000 1:250K 1:250K 40 T308  1:500 1:50K 1:50K 400 T34  1:5001:250K 1:250K 400 P792  1:25K 1:50K 1:100K 400 A32  1:5000 1:25K 1:250K

The HIV647-βgal elicited antibodies that recognized specifically notonly HIV647-βgal, but also the twenty-two amino acid HIV647 peptide. Theresults show that the HIV647 epitope is very immunogenic and βgal is apotent immunocarrier in monkeys. Specific immune response to the HIV647epitope was obtained when as little as 4 μg of the fusion protein,equivalent to only 80 ng of the HIV sequence, is used. In immunodots thetiter of anti-HIV647 antibodies was 1:64000, which was only fivefoldlower than the titer obtained against the large (400,000-dalton) βgaltetramer.

No suppression of the immune response was observed when 4 to 400 μg wasused. Titers to βgal reached a level of 1:250000, independent of thedose. The boost with soluble antigen was most significant at the lowestdose. Also, a somewhat higher anti-HIV647 antibody titer was obtained atthe lowest dose of βgal.

EXAMPLE 7 Immunization of HIV647-βgal Preimmunized M. mulatta withHIV582-βgal

Three Rhesus monkeys from Example 6, T313, 027A and T324, were immunizedwith 4 μg of HIV582-βgal, according to the protocol of Example 6. Theantibody titers to both HIV647-βgal and HIV582-βgal as measured byimmunodot are shown in Table 8.

TABLE 8 Antibody response HIV647-βgal and HIV582-βgal. HIV647-βgal TiterHIV582-βgal titer Animal Day 51 Day 147* Day 190 Day 147* Day 190 T3131:64K 1:16K  1:16K <1:100  1:8000 O27A 1:64K 1:8000 >1:16K <1:100 >1:16KT324 1:32K 1:1000  1:2000 <1:100  1:1000 *Day 147 is day 0 forimmunization with HIV582-βgal

The results show that HIV582 is also an immunogenic epitope in Rhesusmonkeys. Preimmunization with HIV647-βgal did not prevent effectiveimmunization with HIV582-βgal. The moderate increase in HIV647 titerindicates that the use of the same immunocarrier in consecutiveimmunizations of two different HIV epitopes helps memory cells generatedfrom the first immunization.

EXAMPLE 8 Immunization of M. mulatta with SIV-βgal Fusion Proteins

Three groups of monkeys (3 animals/group), free of antibodies to bothHIV and SIV, were used. Each group was immunized according to theprotocol of Example 6. In each case 4 μg of antigen was used in theimmunization. One group (animal 3X7, 4GP and 4GI) was immunized with amixture of the four different SIV-βgal polypeptides (SIV88, SIV500,SIV582 and SIV647). A second group was immunized with SIV647-βgal (4GN,4GJ and 4GG) and a third group was immunized with βgal. As in Example 6,antibody titers were determined with both immunodot and ELISA. ELISA wasperformed, as described in Example 6, except that plates were coatedwith synthetic SIV peptides conjugated to BSA.

On day zero and day 14, no sera had detectable antibodies to any SIVepitope at a serum dilution of 1:100. On day 36, all titers wereone-quarter to one-half the values on day 43, which are reported inTable 9. The antibody response to βgal is reported in Table 10.

TABLE 9 Antibody response to SIV epitopes in M. mulatta on day 43.Animal Assay SIV88 SIV500 SIV582 SIV647 3X7 Immunodot 1:256K 1:64K 1:128K 1:256K ELISA 1:16   1:512  1:1024  4GP Immunodot 1:256K 1:64K1:64K  <1:256K  ELISA 1:256   1:1024 1:512   4GI Immunodot 1:64K  1:16K1:32K  1:256K ELISA 1:256   >1:1024  1:256   4GN Immunodot — — — 1:128KELISA — — — 1:1024  4GJ Immunodot — — — 1:256K ELISA — — — >1:1024   4GGImmunodot — — — 1:256K ELISA — — — 1:128  

TABLE 10 Antibody response to the βgal immunocarrier. Immunodot TiterAnimal Day 0 Day 14 Day 36 Day 43 3X7 <1:100 1:1000 1:400K ≧1:400K 4GP<1:100 1:100 1:200K ≧1:400K 4GI <1:100 1:25K 1:400K ≧1:400K

The results demonstrate that all four SIV epitopes are immunogenic inRhesus monkeys when presented as βgal fusion proteins. The immunodot andELISA results show that antibodies were directed exclusively against theSIV moiety of the SIV-βgal. The ability of the anti-SIV-βgal monkeyantibodies to react with SIVmne antigens was tested in western blots ofwhole disrupted virus and with ³⁵S-methionine labelled SIVmne celllysates in a RIP assay. It was found that the anti-SIV-βgal antibodiesreacted with both the transmembrane (p32) and the envelope (gp110) ofSIV. These results clearly demonstrate that presentation of SIV epitopesvia βgal can elicit an immune response which mimics that of native virusantigens. However, none of the immune sera had any neutralizing activityagainst SIV virus in vitro.

The antibody titers for all SIV antigens ranged from 1:16000 to1:256000. Considering that the SIV epitope constitutes only 1-2% of themolecular weight of the βgal immunocarrier, the titer level isexceptionally high. Antibody titers to the immunocarrier areapproximately 1:400000.

The natural antibody response in monkeys infected with SIVmne are:

SIV88  <1:100-1:400  SIV500 1:100-1:1600 SIV582 1:25000-1:400000 SIV6471:400-1:6400

Thus, with the exception of SIV582, the immunization with all SIV-βgaltested elicited an antibody response 10-100 fold higher than thatelicited against these epitopes by the virus itself.

Immunization with a combination of all four SIV-βgal fusion proteins didnot produce antigenic competition. The level of specific antibodies toSIV647 was comparable when immunization was with SIV647-βgal alone or incombination with other fusion proteins.

EXAMPLE 9 Challenge of SIV-βgal Immunized M. mulatta with SIVmne

The three monkeys immunized with the combination of all four SIV-βgalfusion proteins (3X7, 4GI and 4GP) were boosted at day 290. The boostwas performed with soluble antigen. Each monkey received 40 μg of eachof the SIV-βgal peptides by intramuscular injection.

Twenty-one days after the boost, the monkeys were challenged with viableSIV. All three immunized monkeys developed serologic evidence oftransient SIV infection, whereas all three control monkeys developedpositive western blots to numerous viral antigens, had reversetranscriptase activity detectable in their sera on repeated occasions,and were positive for viral nucleic acid sequences, indicating thepresence of ongoing SIV infection. The results are summarized in Table11.

TABLE 11 Results of reverse transcriptase and western blot assays onsera of monkeys challenged with SIVmne. Immunized monkeys ControlMonkeys 3X7 4GI 4GP 4GC 4HS 3XP Day RT WB RT WB RT WB RT WB RT WB RT WB−6 − − − − − − 0 − − − − − − 7 − − − − − − 14 − ND ND ND ND ND 21 − − −+(2) − − − ND − − − 27 − + − + + + 41 − ±(1) + ++(2) + ++(3) − − − − +++(3) 55 − − − + − + 64 − − − ++(4) − ±(3) + +++(4) − ++(4) nd ++(3) 94− − − + − − Day zero = SIVmne challenge day RT - Cocultivation-ReverseTranscriptase assay WB - Western blot performed on SIVmne partiallypurified lysate (1) env p32 (2) env p32 and p28 (3) env p32 and gpll0 aswell as p28 (4) env p32 and gpll0 as well as p28 and p16

One of the three immunized monkeys (3X7) never became virus positive andtwo were transiently positive. Unlike the controls, none of the threeexperimentally vaccinated animals showed evidence of infection (by RT)55 days post challenge. Two of the three developed a booster responseagainst only the antigens with which they were immunized, demonstratingthat post-vaccination exposure to the virus was capable of inducing ananamnestic booster response to the vaccine.

The antibody titer for the monkeys was determined by ELISA. All sampleswere run in duplicate at a 1:400 dilution and read on a V_(max) ELISAreader at 850 nm. The results, expressed as mOD/min by a kinetic read,are summarized in Table 12.

TABLE 12 Antibody titer of monkeys challenged with SIVmne. Days postchallenge with SIVmne Animal 1 14 21 28 41 55 84 118 148 174 4GI 1.7 1.1nd 10 9.2 8.0 6.4 7.1 5.8 5.8 4GP 0.1 0.1 nd 0.9 6.0 0.8 1.1 1.7 1.8 1.63X7 0.2 0.2 nd 6.5 5.2 2.9 1.0 1.0 1.0 0.7 4GC 0.1 0.1 0.6 3.1 1.4 8.615 12 13 14 4HS 0.1 0.1 0.6 2.4 5.2 6.8 8.5 8.8 10 11 3XP 0.1 0.1 1.03.4 6.0 7.1 12 12 20 23

In all the monkeys there was an increase in antibody titer(crossreacting with HIV-2). In immunized monkeys, antibody leveldecreased between day 28 and day 41 in 3X7, between day 41 and day 55 in4GP, and between day 118 and day 146 in 4GI. In control monkeys, thereis a progressive increase in antibody level. The decline in antibodylevel indicated again that in monkeys immunized by SIV-βgal, SIV isbeing eliminated.

The protection of vaccinated monkeys against infection was quitesurprising in view of the fact that SIV-βgal did not induce neutralizingantibodies in vitro. This finding is consistent with the clinicalobservation that actively infected and ill AIDS patients have highlevels of such neutralizing antibodies. The highly conserved antigenicsequences result in antibodies that are not neutralizing in vitro butare protective in vivo. Because the absolute level of antibody does notexhibit a straightforward correlation with protection against SIVinfection, it is possible that cellular immune responses may be inducedby these vaccine constructs and may play an important role in protectionagainst SIV infection in vivo.

What is claimed is:
 1. A fusion protein comprising: an amino acidsequence selected from the group consisting of NVTENFNMWKN,KAKRRVVQREKRAVG, and EESQNQQEKNEQELLELDKWA; and a non-HIV polypeptidesequence, wherein said amino-acid sequence and said polypeptide sequencecomprise the backbone of said fusion protein, wherein said fusionprotein reacts with an HIV-positive serum.
 2. A fusion protein asclaimed in claim 1, wherein the amino acid sequence is NVTENFNMWKN.
 3. Afusion protein as claimed in claim 1, wherein the amino acid sequence isKAKRRVVQREKRAVG.
 4. A fusion protein as claimed in claim 1, wherein theamino acid sequence is EESQNQQEKNEQELLELDKWA.
 5. A fusion protein asclaimed in claim 1, wherein the non-HIV polypeptide sequence possessesan enzymatic property.
 6. A fusion protein as claimed in claim 1,wherein the non-HIV polypeptide sequence is one to which most humans donot have any antibodies.
 7. A fusion protein according to claim 1,wherein said amino acid sequence is joined via a peptide bond to anN-terminus of said non-HIV polypeptide sequence.
 8. A fusion protein asclaimed in claim 1, wherein the amino acid sequence is NVTENFNMWKN.
 9. Afusion protein as claimed in claim 1, wherein the amino acid sequence isKAKRRVVQREKRAVG.
 10. A fusion protein as claimed in claim 1, wherein theamino acid sequence is EESQNQQEKNEQELLELDKWA.
 11. A fusion proteinaccording to claim 7, wherein said non-HIV polypeptide sequence isβ-galactosidase.
 12. A fusion protein according to claim 1, wherein saidnon-HIV polypeptide sequence is β-galactosidase.
 13. A fusion proteinaccording to claim 1, wherein said amino acid sequence is one ofNVTENFNMWKN and KAKRRVVQREKRAVG.